Extra-coding features of mRNA are essential for hERG channel function This hERG potassium channel plays an essential role in cardiac repolarization, and malfunction of this channel caused by mutation leads to Long QT Syndrome, type 2 (LQT2). LQT2 results in delayed repolarization of the cardiac myocyte, leading to ventricular arrhythmias, syncope and sudden death. There are over 600 documented mutations in KCNH2 associated with Long QT syndrome and these mutations occur throughout the length of the gene, without definitive mutational hotspots. Little investigation has been focused on what causes the hERG channel to be intolerant to mutation. Our hypothesis is that extra-coding features on the mRNA level, such as GC content, rare codon usage and mRNA structure contribute to determining correct protein synthesis of the hERG channel, and that mutational changes that disrupt these features lead to Long QT Syndrome. Previous studies from our lab support this hypothesis. HERG-CM, a codon modified version of the hERG mRNA, was made with reduced GC content (51%) and decreased use of rare codons, while maintaining the exact amino acid sequence. hERG-CM protein was translated and trafficked differently than the native hERG (hERG-NT) channel. To further understand the mechanism behind these observations, Ribosomal Profiling will be used to investigate differences in ribosomal movement across the hERG-NT and - CM mRNA. Capillary Electrophoresis will also be used to investigate structural differences between these two mRNAs. We hypothesize that the sites of ribosomal pausing, caused by rare codons or mRNA structure, allow for important secondary and tertiary structure formation and trafficking of the HERG protein. These pausing locations in the hERG-NT and -CM mRNAs will be investigated, and correlated with known LQT2 mutations to investigate their impact on Long QT pathogenesis. The influence of these extra-coding features of the mRNA on protein folding will also be investigated, through pulse proteolysis, to examine differences in intermediate or final folded structure. hERG-CM and hERG-NT protein function will also be assessed through electrophysiology. Successful completion of this project will provide novel information on how mRNA impacts not only the primary structure of a protein, but also the folding intermediates, translational efficiency, trafficking efficiency, and function of the protei product. Ongoing studies will have implications for evaluation and treatment of hereditary and acquired arrhythmias.